We propose to continue our study of the genetics, physiology, and biochemistry of the NRI-NRII two- component signal transduction system that plays a major role in controlling nitrogen assimilation in E. coli. This system provides a paradigm for the regulation of central metabolism by signals of carbon and nitrogen status. Our work on the NRI/NRII system also provides a paradigm for understanding two-component signal transduction systems. Two component signal transduction systems are the most common type of signal transduction system in bacteria and are also present in lower eukaryotes and plants. In bacteria, these systems control the cell cycle, development, virulence, chemotaxis, numerous responses to environmental stress, and various aspects of metabolism. Our work will also provide a paradigm for signal transduction by PII proteins, which are the most widely distributed signal transduction proteins in nature, Our studies investigate how Pll proteins integrate distinct signals and control receptors that are signal-transduction enzymes. Thus, the proposed research will directly investigate signal transduction mechanisms that are widely-occuring in nature and have significant impact on human health and well-being. The proposed work will focus on structure/function studies of the NRI and NRII proteins of E. coli. The approaches use a variety of genetic, biochemical, and biophysical methods and are designed to provide detailed information on the mechanisms of regulation. The Specific Aims include: (1) Determining the mechanism of regulation of NRII kinase and phosphatase activities by Pll, and (2) Determining the mechanism of the NRI "autophosphatase" activity, and its control by the complex of NRII and Pll. In both Specific Aims, biochemical approaches are used to identify interacting surfaces of the signalling proteins, the chemistry of the activities, and the interactions of protein domains that result in regulation of the catalytic activities. Genetic approaches are used to define the functions of the proteins, and map specific functions to regions of proteins. Biophysical approaches are used to determine the structures of proteins. Given their widespread occurance in nature and direct link to numerous public health and agricultural issues, understanding the mechanisms of signal transduction used by two-component systems and Pll signalling proteins is an important objective. Our studies with a model system should allow rapid progress.